Tachycardiomyopathy entails a dysfunctional pattern of interrelated mitochondrial functions

Tachycardiomyopathy is characterised by reversible left ventricular dysfunction, provoked by rapid ventricular rate. While the knowledge of mitochondria advanced in most cardiomyopathies, mitochondrial functions await elucidation in tachycardiomyopathy. Pacemakers were implanted in 61 rabbits. Tachypacing was performed with 330 bpm for 10 days (n = 11, early left ventricular dysfunction) or with up to 380 bpm over 30 days (n = 24, tachycardiomyopathy, TCM). In n = 26, pacemakers remained inactive (SHAM). Left ventricular tissue was subjected to respirometry, metabolomics and acetylomics. Results were assessed for translational relevance using a human-based model: induced pluripotent stem cell derived cardiomyocytes underwent field stimulation for 7 days (TACH–iPSC–CM). TCM animals showed systolic dysfunction compared to SHAM (fractional shortening 37.8 ± 1.0% vs. 21.9 ± 1.2%, SHAM vs. TCM, p < 0.0001). Histology revealed cardiomyocyte hypertrophy (cross-sectional area 393.2 ± 14.5 µm2 vs. 538.9 ± 23.8 µm2, p < 0.001) without fibrosis. Mitochondria were shifted to the intercalated discs and enlarged. Mitochondrial membrane potential remained stable in TCM. The metabolite profiles of ELVD and TCM were characterised by profound depletion of tricarboxylic acid cycle intermediates. Redox balance was shifted towards a more oxidised state (ratio of reduced to oxidised nicotinamide adenine dinucleotide 10.5 ± 2.1 vs. 4.0 ± 0.8, p < 0.01). The mitochondrial acetylome remained largely unchanged. Neither TCM nor TACH–iPSC–CM showed relevantly increased levels of reactive oxygen species. Oxidative phosphorylation capacity of TCM decreased modestly in skinned fibres (168.9 ± 11.2 vs. 124.6 ± 11.45 pmol·O2·s−1·mg−1 tissue, p < 0.05), but it did not in isolated mitochondria. The pattern of mitochondrial dysfunctions detected in two models of tachycardiomyopathy diverges from previously published characteristic signs of other heart failure aetiologies. Supplementary Information The online version contains supplementary material available at 10.1007/s00395-022-00949-0.


Animal model of tachycardiomyopathy
For in vivo investigation of tachycardiomyopathy, an animal model was used as previously described [1]. Male New Zealand White rabbits were housed under standard conditions with regular, unrestricted diet. For pacemaker implantation, anaesthesia was induced by xylazine 5 mg/kg and ketamine 50 mg/kg i.m. and maintained by continuous i.v. administration (xylazine 1.5 mg/kg and ketamine 15 mg/kg) under close monitoring of the animal's vital signs.
A custom-made, 2 Fr. unipolar pacemaker lead (DMTpe, Nufringen, Germany) was inserted into the right internal jugular vein and fixed into the right ventricular apex under fluoroscopic guidance. A programmable cardiac pacemaker (Advisa DR MRI SureScan, Medtronic, Minneapolis, MN, USA) was implanted subcutaneously into the right abdominal wall and connected to the subcutaneously tunnelled pacemaker lead. In the early post-surgery period, carprofen 4 mg/kg and enrofloxacin 5 mg/kg were administered subcutaneously for analgesia and antibiotic prophylaxis. After recovery of at least 10 days, incremental tachypacing was conducted by programming a V00 mode with a cycle length of 182 ms (330 bpm) for ten days, followed by 167 ms (360 bpm) for ten days and 158 ms (380 bpm) for further ten days. After completion of tachypacing, echocardiographic assessment was performed under moderate sedation (xylazine 1 mg/kg i.m. and ketamine 10 g/kg i.m.) and temporarily intermitted pacing

Chronic in vitro electrical field stimulation of human induced pluripotent stem cell cardiomyocytes
Human induced pluripotent stem cell cardiomyocytes (iPSC-CM) were differentiated from four healthy individuals as previously described [2]. Somatic cells were reprogrammed using nonintegrating systems, followed by differentiation into iPSC-CM by Wnt modulation and metabolic selection. Purity of iPSC-CM was confirmed by flow cytometry (~90% cardiac TNT+), cardiac immunofluorescence, morphology, and qPCR for cardiac sub-type marker (data not shown). After cultivation for 90 days, cells were transferred into 6-well plates equipped with C-Dish carbon electrodes connected to a C-Pace EM electrical stimulator (both IonOptix, Westwood, MA, USA). To simulate persistent tachycardia, iPSC-CM cultures were subjected to chronic electrical field stimulation with 120 bpm for 24 hours (early TACH) or seven days (TACH). IPSC-CM paced with 60 bpm for 24 hours or seven days served as control groups (early CTRL or CTRL, respectively). Effective capture during field stimulation was verified by light microscopic visualization of cell contraction. To account for variation between cell differentiation experiments, chronic in vitro stimulation of iPSC-CM and subsequent experiments were carried out in a paired design. The study was approved by the ethical committee of the University of Göttingen, Germany (ref. no. 10/9/15).

Transmission Electron microscopy
Transmission Electron Microscopy (TEM) was performed to analyse ultrastructural changes in cardiomyocytes with focus on mitochondrial distribution in the cell. For this purpose, rabbit left ventricular (LV) tissue samples were fixed in Karnovsky-fixative (0.1M cacodylate-buffer with 2.5% glutaraldehyde and 2% paraformaldehyde) for at least 48 hours, followed by postfixation with 1% osmium tetroxide at pH 7.3. Samples were then dehydrated in graded ethanol, embedded in EMbed-812 epoxy resin (Science Services, Munich, Germany) and finally polymerized for 48 hours at 60°C into an EPON block. Semithin sections of 0.75µm thickness were cut and stained with toluidine blue and basic fuchsine. After selection of appropriate areas of interest, the EPON block was trimmed, and ultrathin sections (80nm thickness) were cut on a Reichert Ultracut-S ultramicrotome (Leica, Wetzlar, Germany). Sections were mounted on grids and stained with aqueous 2% uranyl acetate and lead citrate solution for 10 min each. IPSC-CM samples were cultured in 6-well-plates. The cell culture medium was discarded and replaced with Karnovsky-fixative for at least 6 hours. Afterwards, the cells were scraped and pelletized at 200g. The cell pellets were then enclosed with cytoblock (Epredia, Portsmouth, NH, USA), then with 4% low melting agarose (Thermo Fisher Scientific). The further handling was the same as for the tissue samples. The sections were analysed by means of a LEO912AB electron microscope (Zeiss, Jena, Germany) operated at 100kV. Images were taken by a side-mounted 2k x 2k-CCD-camera (TRS, Moorenweis, Germany).

Hydroxyproline content
To evaluate collagen content in rabbit LV, hydroxyproline concentration in homogenized tissue was quantified using a commercially available colorimetric assay kit (ab222941, Abcam) according to the manufacturer's instructions.

Mitochondrial and tissue redox state
To determine mitochondrial redox state NADH/NAD + in rabbit LV tissue, a commercially available fluorometric assay kit (ab176723, Abcam) was used according to the manufacturer's instructions. Tissue redox state GSH/GSSG in rabbit LV was quantified as previously described [3]. In brief, total glutathione was determined in homogenized tissue by measuring
Fibres were mechanically separated using pointed forceps in a petri-dish in BIOPS on ice, permeabilization was controlled by microscopy. Samples were subsequently permeabilised by gentle agitation for 30 min at 4°C in BIOPS containing 50 μg/mL of saponin followed by a washing step in MiR05 for 10 min at 4°C by gentle agitation. Afterwards, wet weight of the biopsies was determined and samples were immediately transferred to the oxygraph chambers for measurement of mitochondrial respiration.
Preparation of iPSC-CM cultures 2.5*10 6 cells/ml were harvested, resuspended in mitochondrial medium MiR05, and immediately transferred to the oxygraph chambers for determination of mitochondrial respiration.

Determination of mitochondrial respiration
Activity of the respiratory system was analysed in a two-channel titration injection respirometer (Oxygraph-2k, Oroboros, Innsbruck, Austria) at 37°C. In principle, the same protocol was applied to iPSC-CM cultures and to myocardial biopsies; however, cultured cells were measured under normoxic conditions from air saturation to 50 µM, whereas for cardiac muscle an oxygen range from 400 µM to 200 µM was used to avoid oxygen limitation due to diffusion. Oxygen levels were increased by the addition of H2O2 to a catalase (280U/ml) containing MiR05. The substrate inhibitor titration protocol (SUIT) was only in the beginning slightly different between cultured cells and cardiac muscle biopsies. In iPSC-CM, after a -7 -stabilization phase of 25 min, ROUTINE respiration of intact cells was measured, subsequently malate was added, and the plasma membrane was permeabilized with digitonin (16.2 µM).
For myocardial biopsies, malate was added immediately after fibres (already saponin permeabilized) had been transferred to the Oxygraph chambers followed by a stabilization phase of 10 to 20 min. In both set-ups, complex I activity was stimulated by the following substrates: pyruvate (5 mM), ADP (5 mM), and glutamate (10 mM). Capacity of the oxidative phosphorylation system was determined by convergent electron flow through complex I and II after addition of succinate (10 mM). Subsequently, capacity of the electron transport system was measured by uncoupling with p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCP, 1.5 µM), injected stepwise to avoid inhibitory effects [33]. After rotenone addition (0.5 µM), maximum capacity of complex II was measured. Finally, residual oxygen consumption was determined after myxothiazol addition (0.5 µM).
Quality of muscle fibre preparation based on integrity of the outer mitochondrial membrane was checked by the addition of cytochrome c (10 µM). Since cytochrome c had no stimulatory effect, integrity of the outer mitochondrial membrane could be concluded.

Determination of citrate synthase activity
Activity of the mitochondrial marker enzyme citrate synthase (CS) was determined in iPSC-CM cultures. After finishing the SUIT protocol, the total volume of medium containing suspended iPSC-CM was taken directly from the oxygraph chamber, shock frozen, and stored at -80°C.
After thawing, specific CS activity (IU*ml -1 ) in the samples was quantified photometrically measuring the conversion of DTNB to TNB at 412 nm coupled to the CS-catalysed reaction of acetyl-CoA and oxalacetate to citrate. For photometry, a NanoDrop 2000c (ThermoFisher Scientific) was used.

Normalization of respiration rates
Respiration rates were calculated as the time derivative of oxygen concentration per chamber volume (pmol*s -1 *ml -1 ). For heart muscle fibres, respiration rates were normalized to wet tissue weight (pmol*s -1 *mg -1 ). For iPSC-CM, respiration was normalized to CS activity (pmol*s -1 *IU -1 ).

High-resolution respirometry of isolated mitochondria
Freshly isolated cardiac mitochondria from rabbit LV were used for high-resolution respirometry measurements that were performed as described previously [44]. Oxygen consumption was assayed at 37°C with an Oxygraph-2k high-resolution respirometer and DatLab software was used for data acquisition and analysis (Oroboros Instruments). Two mitochondrial respiration protocols were used to quantify oxygen consumption rate upon supplementation of pyruvate and glutamate (for carbohydrate metabolism) or fatty acids (for β-oxidation) as a fuel. In the "carbohydrate" protocol, measurements of complex I and II activity were performed with 5 mM each of pyruvate, glutamate, malate, and 10 mM succinate. The metabolites were added as reduced substrates after initially recording residual oxygen consumption resulting in leak respiration, followed by increasing concentrations of ADP (0.03, 0.1, 0.3, 1 mM). In the "fatty acid" protocol, respiration was measured using 1 mM carnitine, 3 mM malate, 10 µM palmitoyl-CoA, and 10 µM oleoyl-L-carnitine. Finally, oxygen consumption coupled to ADP phosphorylation was inhibited by adding 1.25 µM oligomycin followed by titration with 10 µM DNP to determine ETS capacity. Mitochondrial membrane potential was simultaneously probed using 1 µM TMRM and Smart Fluo-Sensor Green as described before [4].

Mitochondrial hydrogen peroxide emission in rabbit left ventricular tissue
H2O2 emission from isolated mitochondria was measured with Amplex UltraRed (Thermo Fisher Scientific), a sensitive and H2O2-specific indicator that is converted to the fluorescent molecule resorufin (λexc 535 nm; λem 595 nm) upon reaction with H2O2. Oxidation of Amplex UltraRed by H2O2 is catalysed by horseradish peroxidase (HRP). The assay was performed at 37°C using an Oxygraph-2k high-resolution respirometer (Oroboros Instruments) with a Smart Fluo-Sensor Green-Unit as previously described [5].

Mitochondrial calcium retention
Mitochondrial calcium retention in isolated mitochondria from rabbit LV tissue was quantified as described previously [3] with minor modifications using a fluorescence plate reader (Infinite Densitometry was performed using ImageJ (v.1.53g)

Acetylome analysis
For analysis of the mitochondrial acetylome in rabbit LV, sequential window acquisition of all theoretical mass spectra mass spectrometry (SWATH-MS) of isolated mitochondria was conducted as previously described [6]. In brief, mitochondrial isolation of LV tissue was performed by trypsin digestion, dounce homogenization, and sequential centrifugation steps with 600 to 8000 g. Precipitated proteins of mitochondrial isolates of rabbit LV tissue were reduced, carbamidomethylated and digested. 1 μg of the resulting peptide mixtures was subjected to SWATH-MS measurements. The SWATH-library was build using UniProt_TrEMBL entries for rabbit (Version 2021_01) and ProteinPilot (v.5.0, AB Sciex, Darmstadt, Germany), Paragon Method Special Factor: Acetylation emphasis. The library-search was performed as Thorough Search ID to also include missing cleavage sites. For further analysis, the SWATHruns were then processed using PeakView (v.2.2, AB Sciex). Label free data were exported, normalized to total intensity, and filtered either for acetyl-bearing peptides or pairs of modified and unmodified peptides present in both groups SHAM and TCM.

Metabolome analysis
High performance liquid chromatography-high resolution mass spectrometry Samples and blanks (pure H2O), QCs, and UltimateMix (UM) were measured in a stratified randomized sequence and analysed with a Dionex Ultimate 3000 HPLC (Thermo Fisher Scientific) using a reversed-phase Atlantis T3 C18 pre-and analytical column (Waters, Milford, MA, USA) with 10 µl injection volume as previously described [7]. Raw data was converted into mzXML (msConvert, ProteoWizard Toolkit v3.0.5) and detected m/z of known metabolites were identified using PeakScout (developed in-house) using a reference list containing accurate mass and retention times.
All metabolites were controlled for their analytical quality and graded into two classes: (I) suitable for multivariate analysis and univariate analysis (MVA_UVA) and (II) suitable for univariate analysis (UVA). Quality assessment was done as described in Vogel et al [7]. A separate control for outliers due to sequence position, weight, date of sampling, and sample extraction events was conducted via Principal Component Analysis (PCA).
To correct for weight differences and to reduce technical variability, median normalization was performed. Each metabolite was normalized to the sample median by first scaling the peak area and calculating the median. Normalized peak areas were then log-transformed. Waters,) with 10 µl injection volume. Metabolite separation was achieved with a 25 mingradient adapted from Liu et al. [8]. 90% H2O / 10% ACN with 20mM Ammonium acetate (pH 9) was used as eluent A and 10% H2O / 90% ACN with 20mM Ammonium acetate as eluent B.
Electrospray ionization (ESI) was used for positive and negative ionization and masses between 70 and 1050 m/z were detected with a resolution of 140000 in full scan mode.
Conversion of raw data, identification of metabolites, statistical analysis, and quality assessment was done as described above; for threshold details refer to Supplemental

Clinical chemistry
High-sensitivity troponin T in rabbit serum samples was measured using the cobas pro platform (modules c503 and e801, Roche Diagnostics) by an immunoassay (Elecsys Troponin T hs, Roche Diagnostics). Although the assay is only certified for human sample material, we were able to show in a preliminary study that they were also usable for rabbit samples (data not shown).

Natriuretic peptide measurements
To determine BNP gene expression in rabbit LV tissue, mRNA extraction and cDNA synthesis were performed as described above. Real-time RT-PCR was carried out in triplicate on a ViiA7 Real-Time PCR system (Thermo Fisher Scientific) using QuantiTect SYBR Green RT-PCR kit (Qiagen). GAPDH was used as housekeeping gene. To quantify cGMP tissue concentration in rabbit LV, a competitive enzyme immunoassay (cGMP ELISA kit, Cell Biolabs, San Diego, USA) was performed according to the manufacturer's instructions.

Supplementary Tables
Supplementary Table 1

: Expression fold change (TCM/SHAM) of genes of mitochondrial metabolism in pathway-focused transcriptomics of rabbit left ventricular tissue.
Mitochondrial translocases are highlighted in grey.